This project aims to develop protocols that will lead to the establishment of a robust high-throughput pipeline for the atomic-level structural characterization of membrane protein microcrystals grown in membrane-like environment of lipidic cubic phase (LCP) using serial femtosecond nanocrystallography (SFX) at free electron X-ray laser sources (XFELs). With the use of SFX, we will obviate the need for obtaining large crystals, effectively eliminate radiation damage issues through diffraction before destruction (i.e., diffraction data are collected prior to onset of any damage), simplify handling, as harvesting and freezing are not required, and significantly reduce the time from obtaining initial crystal hits to collecting full data sets. Our long term goal is the integration of this technology into our structural determination pipeline enabling the determination of a large number of three-dimensional structures of G protein-coupled receptors (GPCRs)-ligand complexes addressing questions on ligand selectivity and efficacy using structure-based drug discovery (SBDD) approaches. Our goal will be achieved through the following specific aims. Aim 1: Develop protocols for the production of samples of GPCR-ligand complexes and for the generation, and characterization of large number of microcrystals that can be used for SFX studies. Aim 2: Develop protocols for SFX data collection, processing and structure solution of GPCR-ligand complexes. Aim 3: Integrate protocols developed in Aims 1 and 2 into the GPCR Structure Determination Pipeline and optimize and validate the modified pipeline by determining the structure of novel GPCRs including a number of receptor-ligand complexes. GPCRs constitute the largest family of membrane proteins in the human genome with approximately 800 members and are responsible for transmitting variety of extracellular signals inside the cell, thereby controlling all major physiological responses, including vision, olfactory, immune defense, reproduction, digestion, mental behavior and others; several GPCRs are exploited as co-receptors for entry by HIV and other pathogens. GPCR signaling through multiple effector pathways has profound therapeutic implications, which underscores the need to understand the receptor both biochemically and structurally in the proper context. GPCRs are the target of ~40% of currently marketed drugs. However, detailed understanding of their mechanism of action and ligand selectivity is limited by a lack of structural information. The structure determination of GPCRs is hampered by the difficulty of preparing large amounts of homogenous and stable samples and growing sufficiently large crystals for high- resolution structure determination even when using state-of-the art microfocus beamlines at synchrotron sources.